The proposed research has three major goals: 1.) to determine the mechanism of action of botulinum toxin, 2.) to use clostridial toxins as pharmacological tools for studying the autonomic nervous system, and 3.) to develop chimeric toxins for analyzing mechanisms of transmitter storage and release. To determine the mechanism of toxin action, the applicant proposes to isolate and characterize the eight native botulinum toxins, as well as their major fragments obtained by disulfide bond reduction and/or limited proteolysis. Antibodies will be prepared for all toxins and toxin fragments. These molecules will be used in three basic types of experiments, these being: i.) chemical modification of toxins and fragments, ii.) chemical modification of toxin-susceptible membranes, and iii.) toxin directed (e.g., type specific antibodies) or tissue directed (e.g., pharmacological antagonists) drug interaction studies. To use botulinum toxin as a pharmacological tool, the applicant will employ the isolated toxins and their putative binding fragments in three types of situations, these being: i.) localization and quantitation of postganglionic parasympathetic nerve endings, ii.) production of functional denervation between parasympathetic nerves and effector organs, particularly the heart, and iii.) determination of membrane characteristics that may distinguish various subpopulations of cholinergic nerve ending membranes. To develop chimeric toxins that could be used as probes for nerve ending function, the applicant will attempt to synthesize several novel compounds. Each of these novel substances will have two components, one of which is a ligand that binds to specific types of cell surface receptors, and the other of which is a pharmacologically active substance that has an intracellular substrate. By creating the appropriate hybrid molecule, the applicant should be able to inactivate virtually any cytoplasmic process in nerve endings.